1. Field of the Invention
The present invention relates to a novel endonuclease enzyme which is secreted from immune cells and recognizes bacterial DNA as foreign substance and processes it to generate approximately 10 bases single-stranded oligonucleotide including CpG motif known to involve immune response. In addition, the present invention relates to an immune adjuvant comprising the said single-stranded oligonucleotides of approximately 10 bases generated by the said endonuclease enzyme.
2. Description of the Prior Art
Mammalian animals develop immune systems to defend against foreign agents. The immune systems is classified into natural (nonspecific) immunity or acquired (antigen-specific) immunity. The innate or nonspecific immunity is a primary resistance against diseases caused by one species, and creates defence barrier as four types such as structural, physiological, endocytic and phagocytic, and inflammatory response. Representative examples of the structural defence barrier include skin and mucus. The physiological defence barrier include, for example, temperate, pH, oxygen pressure, and various aqueous soluble factors. The endocytic and phagocytic defence barrier refers to endocytosis and phagocytosis degradation systems in which foreign macromolecules are incorporated into and subsequently degraded by certain cells. The inflammation defence barrier is an inflammatory response which is evolved by various vasoactive and chemotactic agents generated by penetration of bacteria and followed by skin damage. Then, enzyme systems such as clotting, kinin, fibrinolytic or complement are activated. The acquired immunity is different from the innate immunity in that the former possesses specificity, diversity, memory and self and/or non-self recognition. The properties of the acquired immunity are derived from the humoral and cellular immunities which respond by B lymphocytes, T lymphocytes, antibody, cytokine, etc.
The immune response by penetration of microorganisms is generated by the innate mechanism which rapidly recognizes certain molecules of the microorganisms at the initiation stage of the penetration. The proteins and lipids present in microorganisms are well known as agents inducing immune systems which specifically respond to antigen. LPS, formyl methionine, lipoarabinomannan, peptidoglycan, etc. are well known as agents which directly activate the complement system (Maniack, P., and Kapple, J. W. (1994) Cell 76 323-332). Recently, it has been uncovered by many researchers that in mammalian animals, humoral and cellular immunities are activated by distinguishing their intrinsic DNA from bacterial DNA and recognizing the bacterial DNA as foreign agent and that also such bacterial DNA involves innate immunity.
From the fact that a great quantity of anti-DNA antibody is generated during systemic lupus erythematosus (SLE), autoimmune disease, DNA has been investigated in view of antigen or autoantigen. The anti-DNA antibody is serologically considered to be most important in connection with SLE, and functions as a major mediator involving kidney damage, skin eruption, arthritis, etc. (Tan, E. M. (1989) A Texbook in Rheumatology, 11th Ed. D. J. McCarty, cd. Led and Febiger, Philadelphoa, Pa., 1049; Isenberg, D. A., et al (1997) The role of antibodies to DNA in systemic lupus erythematosus-A review and introduction to an international workshop on DNA antibodies held in London, May 1996 Lupus 6, 290-304; Swaak, A. J. G., et al. (1979) Arthritis Rheum, 22, 226-235; and Isenberg, D. A., et al (1994) Arthritis Rheum. 37, 169-180). These antibodies were shown to bind to structure-determining factor present in ssDNA and dsDNA (Isenberg, D. A., et al (1994) Arthritis Rheum 37, 169-180; Pisetskyi, D. S. (1992) Rheum. Dis. Clin. North Am. 18,437-454: and Shoenfeld, Y., and Isenberg, D. A. (1939) Immunol. Today 10,123-126). Although the cause of SLE has not yet exactly revealed, recent studies strongly demonstrate that DNA antigen is significantly implicated in the diseases (Shlomchik, M. J., et al (1987) Proc. Natl. Acad. Sci. USA 84, 9150-9154; Shlomchik, M. J., et al (1990) J. Exp. Med 171, 265-292; and Tillman, D. M., et al (1992) J. Exp. Med. 176, 761-779). Researchers used normal mouse and autoimmune disease mouse to examine immune response to bacterial DNA (Gilkeson, G. S., et al. (1989) Clin. Immunol. Immunopathol. 51, 1482-1486; Gilkeson, G. S., et al (1993) J. Immunol. 151, 1353-1364; and Gilkeson, G. S., et al (1995) J. Clin. Invest. 95, 1398-1402). Unlike mammalian DNA, bacterial DNA possesses potent immunological properties which activate polyclonal B cell and produce antibodies having specificity in mouse (Gilkeson, G. S., et al (1995) J. Clin. Invest 95, 1398-1402; and Gilkeson, G. S., et al (1991) Clin. Immunol. Immunopathol, 59, 288-300). The activity degree is due to the fact that the base sequence motif present in bacterial DNA is different from the base sequence motif of mammalian DNA and may be recognized as foreign agent, i.e., non-self (Messina, J. P. et al (1993) Cell. Immunol. 147, 148-157,; Krieg, A. M. et al (1995) Nature 374, 546-549; and Halpern, M. D. et al (1996) Cell. Immunol. 167, 72-78). When normal mouse is challenged by bacterial DNA, it produces antibody capable of binding to not only bacterial dsDNA but also mammalian and bacterial ssDNA (Gilkeson, G. S. Et al (1991) Clin. Immunol. Immunopathol. 59, 288-300). However, any autoantibody which is cross-reactive to mammalian dsDNA was not produced. Unlike normal mouse, preautoimmune (NZB X NZW)F1. (NZB/W) mouse challenged by dsDNA produced cross-reactive antibody which is bound to mammalian dsDNA (Gilkeson, G. S., et al (1995) J. Clin. Invest. 95,1398-1402). AS such, when autoimmune disease mouse is immunized with bacterial DNA, the animal has the ability to produce anti-dsDNA antibody which is cross-reactive to mammalian DNA. That is because mistaken tolerance which auto-responding anti-dsDNA B cells produced by bacterial DNA in lack of immune-regulator in NZB/W mouse respond to their intrinsic DNA was taken place and thus pathogenic auto-antibodies responding to not only bacterial DNA but also their own DNA were increased (Whock, M. K. et al (1997) J. Immunol. 158, 4500-4506).
A production of antibody by stimulating and activating B cell with protein antigen is well known as the process in which protein antigen is processed by antigen presentation cell (APC) and is bound to major histocompatibility complex (MHC) to induce a presentation of antigen so that MHC-restricted T cell is activated and the activated T cell secrets cytokine to activate B cell (Parker, D. C. (1993) Annu. Rev. Immunol. 11, 331-360; and Clark, E. A., and Ledbetter, J. A. (1994) Nature 367, 425-428). It is also well known that lipoarabinomannan lipoglycans (LAMs), mycolic acid lipids, processed form of constitutive element of mycobacterial cell wall which are distinct from protein antigen can be presented by hCD1b (Beckman, E. M. et al (1994)Nature 372, 691-694; Bendelac, A. (1995) Science 269, 185-186; Sieling, P. A., at al (1995) Science 269, 227-230; and Prigozy, T. I., et al (1997) Immunity 6, 187) and hCD1c (Beckman, E. M., et al (1996) J. Immunol. 157, 2795-2803). CD1 family is nonpolymorphic cell surface glycoprotein which is encoded at the different sites from MHC molecule. Although CD1-T cell binding has not yet clearly defined, it was apparently suggested that mCD1d1 is recognized by CD8+ and CD4+ T cells (Castano A. R., et al (1995) Science 269, 223-226; Cardell, S., et al (1995) J. Exp. Med. 182, 993-1004) and hCD1b is recognized by CD4− and CD8− T cells (Bendelac. A.(1995) Science 269, 185-186). It is thus assumed that CD1 is involved in presentation of various antigens other than proteins found in pathogenic microorganisms. Many studies reported that DNA is involved in anti-DNA-specific B cell stimulation. Krishnan and Marion showed that immunization of mouse with the combination of DNA and peptide could induce anti-DNA antibody ( Krishnaa, M. R., and Marion, T. N. (1993) J. Immunol. 150, 4948-4957). Accordingly, in light of the fact that anti-DNA antibody is generated during various autoimmune disease, it is important to confirm as to whether activation of B cell for production of anti-DNA antibody depends on MHC-restricted T cell stimulation. Waisman suggested that specific activation of T cell by DNA is involved in DNA presentation by MHC class II molecule (Waisman, A., et al (1996) Cell, Immunol. 173, 7-14). That is, he showed the fact that DNA is bound to MHC class II molecule on APC surface and, as results, T cell can be proliferated specifically by DNA and, on the basis of the fact, proposed that DNA takes a critical role in autoimmune disease. However, there are no further studies and knowledge regarding processing and presentation mechanism of DNA antigen. For instance, the matters as to whether bacterial DNA is processed in APC and presented by MHC molecule as in protein antigen or whether other molecules are involved in such a presentation have not yet been explained.
Many researchers showed that vertebrate animals distinguish their intrinsic DNA from bacterial DNA and thereby the immune cell is activated by the bacterial DNA. The bacterial DNA recognized as non-self by vertebrate animals is characterized by generating nonmethylated CpG dinucleotide at high level. The extraordinary difference between bacterial DNA and vertebral DNA may be summarized as follows. First, bacterial DNA generates CpG dinucleotide of 16 dinucleotides at most level, but vertebral DNA generates ¼ of bacterial DNA. This means that CpG suppression exists in vertebral DNA. Second, methylation frequency of CpG dinucleotide present in bacterial DNA is low. While vertebral DNA shows 80% methylation, methylation of microbial cytosine is hardly found (Bird, A. P.(1995) Trends Genet. 11, 94-100). Thirds bacterial DNA is higher than vertebral DNA in the frequency of flanking two 5′-purines and two 3′-pyrimidines at both ends of CpG dinucleotide (Razin A., and Friedman, J. (1981) Prog. Nucleic Acid Res. Mol. Biol. 25, 33-52). The specific structure of the bacterial DNA called “CpG motif” was reported to activate immune response. That is, the activation of immune cell when two 5′-purines and two 3′-pyrimidines were flanked at both ends of CpG dinucleotide (mitogenic CpGs) is much higher compared to when other bases are flanked at both ends of CpG dinucleotide (non-stimulatory CpGs).
Many researchers used chemically synthesized oligodeoxyribonucleotide (ODN) in order to elucidate activation and action of immune cell by specific base sequence of bacterial DNA. Yamamoto and other researchers showed that bacterial DNA increased lytic activation of NK cell and induced the production of interferon-γ (IFN-γ) (Yamamoto, S., et al (1992) J. Immunol. 148, 4072-4076; Cowdery. J. S., et al (1996) J. Immunol. 156, 4570-4575; and Ballas, Z. K., et al (1996) J. Immunol. 157, 1840-1845). Kuramoto reported that such effects are associated with palindromic base sequence of CpG motif included in bacterial DNA ( Kuramoto, E., et al (1992) J. Cancer Res. 83, 1128-1131; and Kimura, Y., et al (1994) J. Biochem. 116, 991-994). In addition, it was reported that bacterial DNA is bound to DNA-binding protein and induces activation of B cell (Gilkeson, G. S., et al (1989) J. Immunol. 142, 1398-1402; Yamamoto, S., et al (1992) J. Immunol. 148, 4072-4076; Gilkeson. G. S., et al (1989) J. Immunol. 142, 1482-1486; Messina, J. P., et al (1991) J. Immunol. 147, 1759-1764; Field, A. K., et al (1967) Proc. Natl. Acad. Sci USA 58, 1004-1010; and Oehler, J. R., and Herverman, R. B. (1978) Int. J. Cancer 21, 221-220). That is, it is understood that the activation of B cell is promoted by CpG motif which consists of six(6) bacterial bases. Immune response by bacterial infection including B cell activation is characterized by producing immune-regulator cytokine(Van Damme, J., et al (1989) Eur. J. Immunol. 19, 163-168; and Paul, W. E., et al Adv. Immunol. 53, 1-29). It was also reported that CpG motif takes part in section of IL-12 involving cellular immunity and IL-6 involving humoral immunity (Halpern, M. D. et al (1996) Cell, Immunol. 167, 72-78; Yi, A. K., et al (1996) J. Immunol. 157, 5394-5402; and Klinman, D. M., et al (1996) Proc. Natl. Acad. Sci. USA 93, 2879-2883). Cytokines generated therefrom include IL-6 which plays a role in activating T cell and B cell (Uyttenhove, C., et al (1938) J. Exp. Med 167, 1417-1427; Muraguchi, A., et al (1999) J. Exp. Med. 167, 332-344; Le, J. M., and Vilcek, J. (1989) Lab. Invest. 61, 583-602; and Hirano, T., et al (1990) Immunol. Today 11, 443-449), IFN-γ which promotes the function of macrophage to eliminate intra- and extra-cellular pathogenic bacteria (Murray, H. W. (1990) Diagn, Microbial. Infect. Dis. 13, 411-421) and IL-12 which regulates production of IFN-γ and activates NK cell (Trinchieri, G. (1994) Blood 84, 4008-4027; Zhan, Y, and Cheers, C.(1995) Infect. Immun. 63, 1387-1390; and Bohn, E., et al (1994) Infect. Immun. 62, 3027-3032). IL-12 and IFN-γ take an important role to eliminate human pathogenic bacteria by increasing type 1 cytokine (Klinman, D. M., et al (1996) Proc. Natl. Acad. Sci. USA 93, 2379-2983; Zhan, Y, and Cheers, C. (1995) Infect. Immun. 63, 1387-1390; Bohn, E., et at (1994) Infect. Immun. 62, 3027-3032; and Heinzel, F. P., et al (1991) Proc. Natl. Acad. Sci. USA 88, 7011-7015). IL-6 stimulates the production of antibody by promoting growth and differentiation of T cell and B cell by type 2 cytokine (Uyttenhove, C., et al (1938) J. Ex. Med 167, 1417-1427; Muraguchi, A., et al (1988) J. Exp. Med. 167, 332-344; Le, J. M., and Vilcek, J. (1998) Lab, Invest. 61, 588-602; and Hirano, T., et al (1990) Immunol. Today 11, 443-449). Indeed, it was observed that mouse with knockout IL-6 gene was easily infected (Yi, A. K. et al (1996) J. Immunol. 157, 5394-5402; and Libert, C. et al (1994) Eur. J. Immunol. 24, 2237-2242). Thus, bacterial DNA is understood to induce the production of cytokine which is involved in cellular and humoral immunity. Recently, it has been further reported that the proliferation and generation of B cell is led by bacterial DNA (Krieg, A. M. et al (1995) Nature 374, 546-549; Liang, H., (1996) J. Clin. Invest. 98, 1119-1129; and Yi, A. K., et al (1996) J. Immunol. 156, 558-564). Study of Krieg showed that CpG motif present in ODN is essential to induce secretion of IgM while activating and proliferating B cell and that the expression of class II MHC molecule, typical phenomenon occurred when B cell was activated, is increased and cell cycle starts from G0 to G1. According to report of Sato (Sato, Y. et al (1996) Science 273, 352-354), it can be seen that when plasmid DNA including immunostimulatory DNA sequence (ISS) with short CpG motif is transfected into monocyte, amounts of IFN-α, IFN-β and IL-12 are increased. This result indicates that if plasmid including ISS is transfected into bone marrow stern cell, then surrounding macrophage and T cell at activated and in vivo rearrangement of the stern cell may be mistakenly occurred. Thus, vector for somatic or stem cell-replacing therapy should be designed not to include ISS. As contrast, one approach to improve the efficacy of vaccine is to design plasmid DNA so as to include many repetitive ISS.
In order to activate cell, bacterial DNA should be incorporated into the cell. It was found that ODN adsorbed on cell culture vessel fails to activate B cell (Krieg, A. M. et al (1995) Nature 374, 546-549) and that when oligonucleotide was lipofected, the incorporation thereof is increased while the activation of NK cell is greatly increased (Yamamoto, T., et at (1994) Microbiol. Immunol. 38, 831-836). It was also found that there was no significant difference between abilities of oligonucleotides to be bound to cell surface whether or not they include CpG motif (Krieg, A. M. et al (1995) Nature 374, 546-549; and Yamamoto, T., et al (1994) Microbiol. Immunol. 38, 831-836). Bennett showed that DNA incorporated into mononuclear cell was degraded in endosomal compartment ( Bennett R. M., et al (1985) J. Clin. Invest. 76, 2182-2190). Stacey showed that bacterial DNA was bound to transcription factor nuclear factor-kB in macrophage and the expression of TNF-α, IL1-β and plasminogen activator inhibitor-2 mRNA was greatly augmented (Stacey, K. J., et al (1996) J. Immuno. 157, 2116-2122). It was expected that incorporation of oligonucleotides into cell with mediation of receptor on cell surface would be taken place by endocytosis (Bennett, R. M. et al (1985) J. Clin. Invest. 76, 2182-2190). Also, a study was performed by using fluorescent-labelled phosphorothioate oligode oxynucleotides in peripheral blood, bone mellow cell and leukemia cell line (Loke, S. L., et al (1989) Proc. Natl. Acad. Sci. USA 86, 3474-3478; Yakubov, L. A., et al (1989) Proc. Natl. Acad. Sci. USA 86, 6454-6458; Zhao, Q., et al (1996) Blood 88, 1788-1795; Ribeiro J. M., and Carson D. A. (1993) Biochemistry 32, 9129-9136), but any property and mechanism thereof have not yet been defined.
Bacterial DNA has been so far understood to take a critical role in the immune system. It has been known that autoimmune disease SLE is occurred by the generation of anti-DNA antibody by bacterial DNA and that CpG motif of bacterial DNA is incorporated into immune cell to activate the cell and thereby promote secretion of cytokine and IgM. However there is no report as to what mechanism enables such critical bacterial DNA to produce antibody and how oligonucleotide having CpG motif is made in cell.
A novel endonuclease was identified by the inventors from human B-lymphoblastic IM9 cell and 12-O-tetradecanoylphorbol 13-acetate-treated differentiated myelogenous U937 and culture medium thereof using DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE).